Glyceraldehyde‐3‐Phosphate Dehydrogenase(NADP) from Sinapis alba L.

Abstract

Partially purified glyceraldehyde‐3‐phosphate dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings was filtered on Sephadex G‐200 in the presence of NADP(H) and NAD(H). With NADP(H) the enzyme elutes as a dissociated species (Mr∼ 170 000), whereas with NAD(H) it travels as an aggregate in the void volume (Mr≥ 800 000). The NAD‐specific enzyme (EC 1.2.1.12) elutes with an Mr of about 160 000, regardless of whether NADP(H) or NAD(H) is present. If the NADP‐dependent enzyme is first filtered with NADP+ on Sephadex G‐200 and then with NAD+ on Sepharose 6B, only part of the enzyme aggregates during the second filtration. The other part (fraction S) travels as a narrow band slightly ahead of the marker cytochrome c. Refiltration of fraction S with NADP+ and with increasing [NADP+]/[NAD+]ratios on Sepharose 6B demonstrates that this transport retardation is NAD‐dependent and counteracted by NADP. The effect is also eliminated by inorganic cations and is not observed on Sephadex G‐200. Dodecylsulfate/polyacrylamide gel electrophoresis of fraction S before and after affinity chromatography on NADP‐agarose shows that the enzyme is pure after transport retardation on Sepharose 6B. It is active with both NADP and NAD and contains at least three different polypeptide chains with apparent Mr of 39 000, 42 000, and 43 000. The NAD‐specific enzyme is shown to be a tetramer of four 38 000‐Mr subunits. Kinetic investigations show that the binding of NADP(H) and NAD(H) with glyceraldehyde‐3‐phosphate dehydrogenase (NADP) is mutually exclusive. The present data indicate that the NADP‐dependent enzyme, a species structurally distinct from the NAD‐specific enzyme, occurs in two different conformations in the presence of NADP(H) and NAD(H) respectively. The existence of a ‘sticky’ NAD conformer is suggested, which interacts with Sepharose 6B and/or aggregates with other molecules in crude or partially purified extracts.

Expression and Characterization of Pea Chloroplastic Glyceraldehyde-3-Phosphate Dehydrogenase Composed of Only the B-Subunit

“…The activity ratio we found for the pea chloroplastic enzyme was 2.6:l (Anderson et al, 199513). Ratios of NADPH-to NADH-linked activity ranging from 0.1:l to 3.5:l have been reported for the purified chloroplastic enzymes from pea (Schulman and Gibbs, 1968;Melandri et al, 1970;McGowan and Gibbs, 1974;Pupillo and Faggiani, 1979), spinach (Yonuschot et al, 1970;Pupillo and GiulainiPiccarri, 1975;Ferri et al, 1990;Trost et al, 1993), white mustard (Cerff, 1978), beet, buttercup, and Arum italicum (Pupillo and Faggiani, 1979). The somewhat higher NADPH to NADH activity ratio found for the recombinant B-subunit enzyme compared with the native chloroplastic enzyme suggests that the activity ratio of the B-subunit is higher than that of the A-subunit.…”

“…Cerff (1978) also reported that the K, of G-3-P dehydrogenase from white mustard for NADPH is 13-fold lower than that for NADH. Ferri et al (1978) reported that the K, of the enzyme from spinach for NADP(H) is about 5-fold lower than that for NAD(H).…”

“…Similar results were obtained with the recombinant enzyme in crude extracts (data not shown). Like the A 2 B 2 isozyme from chloroplasts (Cerff, 1978(Cerff, , 1979Cerff and Chambers, 1978;Pupillo and Faggiani, 1979;Ferri et al, 1990), the recombinant enzyme composed only of Bsubunit forms aggregates. The BA334-subunit activity peak emerged at the same position as the 146-kD rabbit muscle G-3-P dehydrogenase.…”

Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3-phosphate dehydrogenase from pea and spinach

“…Indeed, in the light of the present results this interpretation would best explain why isoenzymes I and II from mustard and barley are virtually indistinguishable on the basis of tryptic fingerprints, immunological cross reactivity and amino acid compositions [8]. If one assumes that proteolytic cleavage of the C-terminal extension may occur in several steps, this concept would also explain why subunit B in certain plants or after particular purification procedures gives protein doublets [5,9]. Experiments are under way to distinguish at the protein level whether isoenzyme II is the proteolytic product of isoenzyme I (A2B;) or whether it is a separate A 4 tetramer.…”

Properties to two high‐molecular‐mass forms of glyceraldehyde‐3‐phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity

“…In some cases it has been reported to be predominantly active with NADP [l, 2, 51, whereas in others NAD is the preferred coenzyme [3, 41. In the presence of NADP the high-M, enzyme dissociated into low-M, forms predominantly active with NADP [4,6,7]. This could be reversed by NAD which promoted the reassociation of the low-M, forms.…”